Usually, cotton fibers are produced by cultivating a cotton plant of the genus Gossypium and collecting the cotton fibers from the capsules (cotton bolls) formed on the cotton plant. There are many varieties of cotton plant, from which cotton fibers with different fiber characteristics can be obtained and used for various applications depending on their fiber characteristics. Cotton fibers are characterized by various properties among which fiber length, fiber fineness and fiber strength are particularly important. Many previous efforts have been made to improve the characteristics of cotton fibers. Attempted improvements have been mainly focused on fiber length and fiber fineness. In particular, there has been a great demand for longer and finer cotton fibers. The variety of cotton plant known as Sea Island is famous for desired fiber characteristics; however, this variety of cotton plant exhibits a poor yield of cotton fibers, therefore the price of Sea Island cotton fibers is very high. If highly yielding cotton plants with fiber characteristics equal to or better than those of Sea Island cotton can be produced, it will be a great contribution to industry.
The methods for improving the characteristics or yield of cotton fibers can be roughly classified into the following three categories:
1. Variety improvement by cross breeding
This method has been utilized most widely so far. At present, almost all the cultivated varieties of cotton plant were bred by this method. However, much time is needed for this method, and because of a limit to the degree of variability, one cannot expect remarkable improvements in fiber characteristics or in yield of cotton fibers.
2. Treatment with plant hormone
Plant hormones such as auxin, gibberellin, cytokinin and ethylene have been widely applied to field crops or horticultural products. Many reports have hitherto been made with respect to the influence of plant hormones on the fiber production of cotton plants, particularly on the fiber elongation mechanism. It is believed that fiber elongation is induced by gibberellin or auxin but inhibited by abscisic acid (Bhardwaj and Sharma, 1971; Singh and Sing, 1975; Baert et al., 1975; Dhindsa et al., 1976; Kosmidou, 1976; Babaev and Agakishiev, 1977; Bazanova, 1977; DeLanghe et al., 1978). Also Beasley and Ting Amer. J. Bot., 60(2): 130-139(1973)! reported that gibberellin has a promoting effect on the fiber elongation in ovule cultures (in vitro) whereas kinetin and abscisic acid have an inhibitory effect on the fiber elongation.
In a field test (in vivo), when non-fertilized flowers of cotton plants were treated with gibberellin just after flowering, there was found a promoting effect on the fiber elongation to a certain degree; in the case of fertilized flowers, however, no significant promotion was caused by gibberellin treatment (The Cotton Foundation Reference Book, Series Number 1, Cotton Physiology, 369, The Cotton Foundation, 1986).
As to the influence of plant hormones on the yield of cotton fibers was analyzed by MaCarty and Hedin who reported as follows: a field test on commercial plant growth regulators were carried out for a period of from 1986 to 1992. They found only in the field test of 1992 that an increase in fiber yield was observed with a Foliar Trigger (manufactured by Westbridge Chemical Co.) containing cytokinin or with FPG-5 (manufactured by Baldridge Bio-Research, Inc.) containing cytokinin, indoleacetic acid and gibberellin; however, no significant increase in fiber yield was observed in the other years J. Agric. Food Chem., 42: 1355-1357 (1994)!.
As described above, for the purpose of improving the characteristics and yield of cotton fibers, a number of studies and reports have been made on conventional plant hormones such as auxin, gibberellin, cytokinin and abscisic acid; however, no effect has been fully confirmed yet, and it cannot be said that these plant hormones are effective for practical use.
In recent years, much attention has been paid to brassinosteroids as a novel group of plant hormones, and the action of these hormones on various plants has been studied. For the first time, Mitchell, Mandave, et al., discovered brassinolide, which is one of the brassinosteroids, from Brassica napus pollen Nature, 225, 1065 (1970)!, and they confirmed that it has a remarkable effect on the cell elongation in the young buds of kidney bean. As described above, brassinolide is one of the steroid compounds with complicated structure, and many compounds with structural similarities thereto have since been discovered from various plants.
The effects of brassinosteroid when applied to cotton plants, was reported by Luo et al. Plant Physiology Communications, 5: 31-34 (1988)! that the treatment of boll stalks with 0.01 or 1 ppm brassinolide reduced the shedding of young bolls in a field test (in vivo). However, no report has hitherto been made that the characteristics or yield of cotton fibers can be improved by the use of any brassinosteroid.
For callus culture (in vitro), Wang et al. Plant Physiology Communications, 28(1): 15-18 (1992)! reported that the addition of 0.01 ppm brassinolide to MS medium induced the callus formation and embryogenesis in cotton plants. However, no report has hitherto been made that the characteristics or yield of cotton fibers can be improved by addition of a brassinosteroid to a medium used for the ovule culture in the production of cotton fibers.
3. Variety improvement by gene recombination technique
In recent years, gene recombinant technique has made startling progress, and several reports have been made on the successful variety improvement in certain kinds of plants (e.g., cotton, tomato, soybean) by introduction and expression of a particular gene in these plants to confer a desired genetic trait thereon. For example, the following studies have been made on cotton plants: one is to improve insect resistance by introduction of a gene coding for BT toxin (Bacillus thuringiensis produced insecticidal protein toxin), and the other is to improve herbicide (Glyphosate) resistance by introduction of a gene coding for 5-enolpyruvilshikimic acid 3-phosphate synthetase.
If a gene associated with fiber formation and elongation can be introduced into cotton plants and expressed in large quantities, it would become possible to make a remarkable improvement in the characteristics or yield of cotton fibers. Further, the introduction of such a gene in anti-sense form makes it possible to suppress the action of this gene. In other words, it is believed that the characteristics and yield of cotton fibers can be controlled by introducing a gene associated with fiber formation and elongation into cotton plants, followed by large-scale expression or suppression of the gene. The method using such a genetic engineering technique can be expected to find wide applications, e.g., more reliable fiber elongation control as compared with the conventional plant breeding by crossbreeding and screening. For this purpose, a gene associated with fiber elongation, which is greatly expressed in a fiber tissue-specific manner at the fiber elongation stage, must be isolated and identified.
At present, however, the knowledge of fiber elongation in plants from the viewpoint of molecular biology is very limited. Although many studies have been made on the elongation of plant cells, most of the control factors remain unknown and the control mechanisms have not yet been elucidated. This is, for example, because a gene expressed specifically in the fiber elongation stage and also in the elongating fiber tissue is difficult to obtain and examine for its function.
A cotton fiber is composed of a single cell differentiated from an epidermal cell of the seed coat and develops though four stages, i.e., initiation, elongation, secondary cell wall thickening and maturation stages. More particularly, an epidermal cell of the ovule begins cotton fiber elongation just after flowering, and a cotton fiber rapidly elongates and completes elongation in about 25 days after flowering. Then, fiber elongation stops, and a secondary cell wall is formed and grown to become a mature cotton fiber. It seems that the examination of the expression of a gene in the ovule, particularly in the cotton fiber tissue, at the fiber elongation stage in the fiber growth process is an effective means of elucidating the mechanism of fiber elongation.
Several attempts have been made on such an isolation of genes from cotton fibers. Many of the genes actively functioning in the fiber cell are closely similar to those which are present in the leaf, ovule or root. The isolation of a gene expressed preferentially in a cotton fiber tissue was reported by Maliykal E. John and Laura J. Crow Proc. Natl. Acad. Sci. USA, 89, 5769 (1992)!. However, only a few reports have been made on the cotton fiber tissue-specific gene.
In recent years, an effective method for isolating a gene which is specifically expressed in different cells or under the particular conditions, i.e., differential display method, was reported. However, there has been no report on the isolation of a cotton fiber tissue-specific gene using the differential display method.